Recombinant 40 KDa Dermatophagoides farinae allergen

ABSTRACT

The present invention is directed to a recombinant mite allergen obtainable by expression of a mite-body-derived gene, a gene which codes for said allergen, a mite allergen fragment, a polypeptide having an epitope contained in said allergen, an expression vector capable of expressing the gene, a bacterium, yeast or mammalian cell transformed with said expression vector, a method for producing said allergen, and a pharmaceutical composition or a diagnostic reagent for the treatment of mite allergic diseases.

FIELD OF THE INVENTION

The present invention relates to a recombinant mite allergen possessingallergen activity.

BACKGROUND OF THE INVENTION

House dust mites are important as a major cause of allergic diseasessuch as atopic bronchial asthma.

Traditionally, hyposensitization therapy using a causative substance ofallergy has been recognized as the most important basic approach to thetreatment of allergic diseases, and its efficacy has already been wellestablished in the treatment of allergic diseases caused by unavoidableinhaled allergen such as pollenosis, house dust allergy and fungalallergy.

However, this hyposensitization therapy necessitates administration of asafe therapeutic antigen since it involves a risk of anaphylaxis due toa sensitized antigen, and investigations are being made on such asensitized antigen.

With respect to mite allergic diseases, two mite species, namelyDermatophagoides pteronyssinus and Dermatophagoides farinae are reportedas important sources of allergen in house dust [Allerg. Asthma, 10,329-334 (1964); J. Allergy, 42, 14-28 (1968)]. From these mite species,major mite allergens were fractionally separated and identified asglycoproteins (pI 4.6 to 7.2) having a molecular weight of 24 to 28 kDand/or proteins (pI 5 to 7.2) having a molecular weight of 14.5 to 20kD, both of which are contained in mite excretion and/or mite bodies [J.Immunol., 125, 587-592 (1980); J. Allergy Clin. Immunol., 76, 753-761(1985); Immunol., 46, 679-687 (1982); Int. Arch. Allergy Appl. Immunol.,81, 214-223 (1986); J. Allergy Clin. Immunol., 75, 686-692 (1985) andother publications].

However, none of the existing antigens for hyposensitization therapy iseffective and safe.

Mite allergen genes have been cloned; for example, with respect to Der pI (molecular weight: 25,371) and Der p II (molecular weight: 14,131 and17,460), the major allergens of Dermatophagoides pteronyssinus, and Derf II (the molecular weight remains undetermined because the initiationcodon remains unidentified), the major allergen of Dermatophagoidesfarinae, the gene of each major allergen was cloned and its nucleotidesequences were determined [Int. Arch. Allergy Appl. Immunol., 85,127-129 (1988); J. Exp. Med., 167, 175-182 (1988); J. Exp. Med., 170,1457-1462 (1989); Int. Arch. Allergy Appl. Immunol., 91, 118-123 (1990);Int. Arch. Allergy Appl. Immunol., 91, 124-129 (1990); Jpn. J.Allergol., 39, 557-561 (1990); Agric. Biol. Chem., 55, 1233-1238(1991)], and attempts have been made to study mite allergens by geneticrecombination technology.

However, none of the existing mite allergens can serve as a sensitizedantigen.

On the other hand, the diagnosis of mite allergic diseases has beenmostly based on inquiry in combination with skin reaction test using ahouse dust extract and/or mite body extract, with measurements of serumIgE antibody titer (relative value) taken by the RAST (radioallergosorbent test) method, an inhalation provocation test and a nasalmucosal provocation test conducted concurrently in only a few cases. Ithas therefore been very difficult to make direct diagnosis of miteallergic diseases.

It has been the traditional practice to use a house dust extract forhyposensitization therapy for bronchial asthma caused by house dustmites as a specific antigen. However, it is subject to extremelimitation with respect to dose and its therapeutic effect is very low,since its chemical composition is very indefinite and it contains a widevariety of impure substances which may induce anaphylaxis.

Thus, from the viewpoint of efficacy and safety, it is desired todevelop a useful antigen for hyposensitization therapy, and it is alsoexpected that such a high quality antigen for hyposensitization therapywill be supplied stably.

However, it is difficult to stably supply such a safe mite allergen insufficient amounts to ensure the desired effect by a method based onextraction and purification of mite allergen from mite culture becausethis method lacks mass-productivity and is subject to quantitativelimitation.

SUMMARY OF THE INVENTION

The present invention aims at overcoming these drawbacks by providing arecombinant mite allergen which is free of anaphylaxis-provokingimpurities and which serves as a safe and effective therapeutic agentand diagnostic reagent for mite allergic diseases.

Accordingly, it is an object of the present invention to provide arecombinant mite allergen obtained by expression of a mite-body-derivedgene.

It is another object of the invention to provide a gene which codes forsaid mite allergen.

It is still another object of the invention to provide a mite allergenfragment.

It is yet another object of the invention to provide a polypeptidehaving an epitope contained in mite allergen or a polypeptide having anepitope which can be regarded as immunologically identical to saidepitope.

It is also another object of the invention to provide a gene which codesfor the above-mentioned polypeptide having an epitope.

It is also another object of the invention to provide an expressionvector capable of expressing the gene of the invention.

It is also another object of the invention to provide a transformantcarrying an expression vector capable of expressing said gene.

It is also another object of the invention to provide a productionmethod for said recombinant mite allergen.

It is also another object of the invention to provide a new therapeuticagent for mite allergic diseases whose active ingredient is therecombinant mite allergen of the invention.

It is also another object of the invention to provide a new diagnosticreagent for mite allergic diseases whose active ingredient is therecombinant mite allergen of the invention.

It is also another object of the invention to provide a new therapeuticagent for mite allergic diseases whose active ingredient is the miteallergen fragment of the invention.

It is also another object of the invention to provide a new diagnosticreagent for mite allergic diseases whose active ingredient is the miteallergen fragment of the invention.

It is also another object of the invention to provide a new therapeuticagent for mite allergic diseases whose active ingredient is theepitope-containing polypeptide of the invention.

It is also another object of the invention to provide a new diagnosticreagent for mite allergic diseases whose active ingredient is theepitope-containing polypeptide of the invention.

The present inventors made intensive investigations to solve theproblems described above, and found a gene which coded for mite allergenpossessing potent allergen activity from mite bodies. The inventors madefurther investigations based on this finding, and developed the presentinvention.

Accordingly, the present invention relates to:

(1) a recombinant mite allergen containing the partial amino acidsequence shown below (SEQ ID NO:1), obtainable by expression of amite-body-derived gene, ##STR1##

(2) a recombinant mite allergen containing the partial amino acidsequence shown below (SEQ ID NO:2), obtainable by expression of amite-body-derived gene, ##STR2##

(3) a mite-body-derived gene which codes for an allergen active proteincontaining the amino acid sequence described in (1) or (2) above,

(4) a mite allergen fragment containing at least an amino acid sequenceencoded in the region of about 170 bp to about 270 bp, about 270 bp toabout 400 bp, or about 170 bp to about 400 bp from the upstream in thenucleotide sequence shown in FIG. 10,

(5) a polypeptide having an epitope contained in mite allergen or apolypeptide having an epitope which can be regarded as immunologicallyidentical to said epitope,

(6) a gene which codes for the polypeptide described in (5) above,

(7) an expression vector capable of expressing the gene described in (3)or (5) above,

(8) a bacterium, yeast or mammalian cell transformed with the expressionvector described in (7) above,

(9) a method for producing a recombinant mite allergen which comprisescultivating the bacterium, yeast or mammalian cell described in (8)above under conditions which allow their gene to be expressed to producea recombinant mite allergen and subsequently recovering said recombinantmite allergen,

(10) a pharmaceutical composition for the treatment of mite allergicdiseases whose active ingredient is the recombinant mite allergendescribed in (1) or (2) above,

(11) a diagnostic reagent for mite allergic diseases whose activeingredient is the recombinant mite allergen described in (1) or (2)above,

(12) a pharmaceutical composition for the treatment of mite allergicdiseases whose active ingredient is the mite allergen fragment describedin (4) above,

(13) a diagnostic reagent for mite allergic diseases whose activeingredient is the mite allergen fragment described in (4) above,

(14) a pharmaceutical composition for the treatment of mite allergicdiseases whose active ingredient is the polypeptide described in (5)above, and

(15) a diagnostic reagent for mite allergic diseases whose activeingredient is the polypeptide described in (5) above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of protein staining and immunological stainingof the mite allergen fused protein expressed by λ gt11 inserting themite allergen cDNA in the host E. coli Y1090 obtained after SDS-PAGEanalysis followed by blot to a nitrocellulose membrane, in which lane 1shows the results for a molecular marker (β-galactosidase, having amolecular weight of about 130000); lane 2 shows the results for thetotal protein of E. coli Y1090 cells; and lane 3 shows the results ofimmunological staining of the recombinant mite allergen (indicated withan arrow) using a rabbit anti-mite-body antigen serum.

FIG. 2 shows the results of agarose electrophoresis of the cDNA whichcodes for mite allergen.

FIG. 3 shows the construction of the recombinant plasmid pAK1.

FIG. 4 is the restriction enzyme map of the cDNA which codes for miteallergen.

FIG. 5 shows the construction of the recombinant plasmid pAKE1.

FIG. 6 shows the construction of the recombinant plasmid pAEX201.

FIG. 7 shows the results of SDS-PAGE analysis of the mite allergen fusedprotein expressed by pAEX201 in the host E. coli pop2136, in which lane1 shows the results for a molecular marker; lane 2 shows the results forthe total protein of E. coli pop2136 cells carrying the plasmid pUEX2;lane 3 shows the results for the total protein of E. coli pop2136 cellscarrying the plasmid pAEX201; and lane 4 shows the results for theprotein localized in E. coli pop2136 membrane carrying the plasmidpAEX201 (the recombinant mite allergen is indicated with an arrow).

FIG. 8 shows the results of gel filtration and SDS removal for the miteallergen fused protein using a couple of Ultrogel AcA 44 and AG 11A8columns.

FIG. 9 shows the results of titration of washed blood cells from a miteasthma patient with the mite allergen fused protein.

FIG. 10 shows the presence or absence of antigen activity in thedeletion mutants of recombinant mite allergen, in which the fragmentsmarked with ○ possess antigen activity.

FIG. 11 shows the presence or absence of antigen activity in thedeletion mutants of recombinant mite allergen, in which the symbols -, +and +++ indicate the degree of antigen activity in the deletion mutantsa, b, c and d.

DETAILED DESCRIPTION OF THE INVENTION

The recombinant mite allergen of the present invention is obtained byexpression of a mite-body-derived gene. Although the mite species usedfor the present invention are not subject to limitation, house dust mitespecies such as Dermatophagoides farinae and Dermatophagoidespteronyssinus are used.

The recombinant mite allergen of the present invention comprises aprotein containing the partial amino acid sequence shown below (SEQ IDNO: 2) and possesses allergen activity. ##STR3##

Another recombinant mite allergen of the present invention comprises aprotein containing the amino acid sequence shown below (SEQ ID NO: 4),which contains the partial amino acid sequence shown above, andpossesses allergen activity. ##STR4##

The recombinant mite allergen of the present invention may be anyvariant resulting from the substitution, deletion, addition ortranslocation of one or more amino acids in the amino acid sequenceshown above, as long as it possesses allergen activity. Such a variantcan be obtained as a naturally-occurring allelic variant or by inducingmutation at a specific site of DNA by recombinant DNA technology.

The recombinant mite allergen of the present invention may be expressedas a fused protein with another protein. In the present specification, arecombinant mite allergen expressed in a fusion with another protein isalso referred to as a fused recombinant mite allergen in some cases.Although the other protein involved in the fusion is not subject tolimitation, examples thereof include β-galactosidase,glutathione-S-transferase and protein A.

The recombinant mite allergen of the present invention may be a proteinfragment comprising only a region essential to allergen activity, andmay comprise a domain essential to allergen activity.

In the present specification, an active fragment possessing allergenactivity is referred to as a mite allergen fragment. Examples thereofinclude fragments containing at least an amino acid sequence encoded inthe region of about 170 bp to about 270 bp, about 270 bp to about 400bp, or about 170 bp to 400 bp from the translation start in therestriction map shown in FIG. 10.

The recombinant mite allergen of the present invention may be obtainednot only by expressing mite allergen protein alone but also byeliminating the other protein from a fused protein.

In short, the recombinant mite allergen of the present invention is anallergen active protein which substantially contains the above-mentionedamino acid sequence obtained by expression of a mite-body-derived gene.

The recombinant mite allergen of the present invention is exemplified bythe following β-galactosidase-fused recombinant mite allergen expressedin E. coli. The fused recombinant mite allergen expressed in E. coli ispurified by gel filtration chromatography using Ultrogel AcA 44(produced by LKB), anti-β-galactosidase antibody immobilized affinitychromatography and anti-mite-body antibody immobilized affinitychromatography, and possesses the following properties as a recombinantmite allergen.

1) Color and appearance: White.

2) Water solubility: Freely soluble.

3) Molecular weight: About 40000, as estimated by SDS-PAGE using theequation given below.

Molecular weight of fused protein-molecular weight of β-galactosidase

4) Contains the partial amino acid sequence shown below (SEQ ID NO:2),##STR5## or contains the amino acid sequence shown below (SEQ ID NO:4),which contains the partial amino acid sequence shown above (SEQ IDNO:1). ##STR6##

5) Possesses antigenicity. Judged on the basis of the ELISA reactivitywith the specific IgG in mite allergy patient's pool serum, rabbitanti-mite-body serum and rabbit anti-β-galactosidase, and the reactivitywith the above-mentioned antiserum after SDS-PAGE of the expressedprotein followed by blot to a nitrocellulose membrane.

6) Possesses allergen activity. Judged by a histamine release test of amite allergy patient leukocyte based on high performance liquidchromatography.

7) Does not induce anaphylaxis reaction. Guinea pigs are immunized withfused protein or mite allergen protein by a conventional method andobserved for anaphylaxis reaction upon booster immunization.

The mite-body-derived gene of the present invention is obtained bypreparing the mRNA from live mite bodies and synthesizing the cDNA by aconventional method using said mRNA as a template. It codes for anallergen-active protein containing the amino acid sequence describedabove in the molecule. Examples of the DNA which codes for the partialamino acid sequence include the sequence shown below (SEQ ID NO:2).##STR7##

The DNA is also exemplified by the nucleotide sequence shown below (SEQID NO:4), which contains the nucleotide sequence shown above. ##STR8##

This DNA sequence is not homologous to any of the DNA sequences reportedfor the allergens Der p I, Der p II and Der f II.

With respect to the mite-body-derived gene of the present invention, thetotal nucleotide chain length of the cDNA is about 1.2 kbp [determinedby agarose electrophoresis, the length is 1143 bp (about 1.1 kbp) whenthe linker is excluded, but the length of the translated base chain is1026 bp (about 1 kbp), including the termination codon, since expressioncompletes at the termination codon before the linker in actualexpression], and it has the restriction enzyme map shown in FIG. 4.

The expression vector of the present invention is bound so that the geneof the present invention described above is expressed in a transformedbacterium, yeast or mammalian cell. The vector DNA used to construct theexpression vector is not subject to limitation; any widely availablevector DNA can be used, including pUC18, pTV118N (produced by TakaraShuzo), pUEX2 (produced by Amersham), pKK233-2 (produced by Pharmacia)and pMAM-neo (produced by Clontech).

pAK1, an expression vector of the present invention, can be obtained by,for example, digesting the mite allergen cDNA inserted in λgt11 phagewith Kpn I-Sac I and ligating to the plasmid vector pUC18 at the KpnI-Sac I site. Another expression vector pAKE1 can be obtained byligating the EcoR I digestion fragment of pAK1 to pUC18 at the EcoR Isite in the same manner as above. A still another expression vectorpAEX201 can be obtained by ligating the EcoR I digestion fragment ofpAKE1 to pUEX2 (produced by Amersham) previously cleaved by EcoR Idigestion.

The bacterium, yeast or mammalian cell transformed with the expressionvector of the present invention is not subject to limitation, as long asit is capable of expressing the gene of the invention. Examples of suchbacteria include E. coli and Bacillus subtilis. Examples of such yeastsinclude Saccharomyces cereviceae. Examples of such mammalian cellsinclude Chinese hamster ovary (CHO) cells, simian COS cells and mousefibroblasts.

The method for production of recombinant mite allergen of the presentinvention comprises cultivating a bacterium, yeast or mammalian celltransformed with an expression vector which permits the expression ofthe gene of the present invention under conditions which permit theexpression of said gene to produce recombinant mite allergen and thenrecovering said recombinant mite allergen.

The method for production of recombinant mite allergen using an E. colitransformant carrying a fused protein expression plasmid inserting themite allergen cDNA is exemplified as follows:

First, an E. coli transformant carrying a β-galactosidase-fused proteinexpression plasmid inserting the mite allergen cDNA is subjected toshaking culture by a conventional method to the logarithmic phase, andfused protein is inductively synthesized by making a temperature shiftor adding a β-galactosidase inducer while maintaining this logarithmicphase. Examples of E. coli strains used as host cells include E. colipop2136 (produced by Amersham), and the strain transformed withexpression vector is named E. coli pop2136 OL-1 (FERM BP-3497).

After completion of the cultivation, cells are harvested, suspended in abuffer containing serine, cysteine, aspartic acid and metallic proteaseinhibitors and disrupted by ultrasonication. The membrane-localizedprotein in the cell debris is extracted with a buffer containing bothprotease inhibitors such as phenylmethanesulfonyl fluoride,monoiodoacetic acid, pepstatin A and ethylenediaminetetraacetic acid anda detergent such as sodium lauryl sulfate (SDS), Triton X-100 or NonidetP40. The mite allergen-β-galactosidase fused protein obtained from theextract or culture concentrate is purified by, for example, gelfiltration chromatography using Ultrogel AcA 44 (produced by LKB),anti-β-galactosidase antibody immobilized affinity chromatography andanti-mite-body antibody im mobilized affinity chromatography. Theanti-β-galactosidase antibody immobilized carrier is prepared bycovalently binding an anti-β-galactosidase antibody (produced by5Prime→3Prime Inc.) to an activated tresyl carrier such as tresyl GM gel(produced by Kurita Water Industries Ltd.), tresyl Toyopearl (producedby Tosoh Corporation) or tresyl Sepharose (produced by Pharmacia). Theanti-mite-body antibody immobilized carrier is prepared by covalentlybinding a rabbit anti-mite-body antibody to the above-mentionedactivated carrier.

The purified recombinant mite allergen fused protein is digested withprotease and then fractionated by one or more of the known purificationmethods such as gel filtration chromatography, ultrafiltration, ionexchange chromatography, affinity chromatography, hydrophobicchromatography, chromatofocusing, isoelectric focusing and gelelectrophoresis, while monitoring the course of fractionation by ELISAand a mite allergy patient leukocyte histamine release test [Arerugi,37, 725 (1988)]. From the active fraction thus obtained, the proteincomponents originating from β-galactosidase are absorbed and removedthrough an anti-β-galactosidase antibody immobilized affinity column,after which the recombinant mite allergen fragment is purified using ananti-mite-body antibody immobilized affinity column.

The above-mentioned methods can also be used singly or in combination topurify the mite allergen fragment from the digested product of the fusedprotein purified by the method described above with a protease such aspronase, subtilisin, thermolisine or trypsin, or from the decompositionproduct obtained by treatment with a chemical such as cyanogen bromide,2-nitro-5-thiocyanobenzoic acid or hydroxylamine.

The production method for recombinant mite allergen of the presentinvention includes not only direct expression of mite allergen proteinbut also the method in which a fused protein expressed as a fusedrecombinant mite allergen resulting from fusion of mite allergen withanother protein via an intervening sequence is recovered andsubsequently the fused other protein is eliminated.

Examples of the other protein to be fused include β-galactosidase,glutathione-S-transferase, protein A and other proteins which aregenerally known to form a fused protein.

Elimination of the other protein can also be achieved by a known method.For example, when the other protein is β-galactosidase and a part ofcollagen or fibrinogen protein is present between the other protein andthe mite allergen protein, the mite allergen protein is purified by oneor more of the methods described above using collagenase or thrombin toleave the fusion protein.

The mite allergen fragment of the present invention can easily beobtained as a recombinant mite allergen by constructing an expressionvector from the DNA which codes for the amino acid sequence of saidfragment by a conventional method and expressing the DNA in anappropriate host.

Moreover, said mite allergen fragment can be used as a synthetic miteallergen, synthesized by the conventional solid phase synthesis method,singly or in conjugation with an appropriate carrier such as human serumalbumin or sea squirt antigen, which has been confirmed as safe inintracutaneous administration.

Examples of the polypeptide of the present invention, which has anepitope contained in the mite allergen derived from mite body, or thepolypeptide having an epitope which can be regarded as immunologicallyidentical to said epitope, include the polypeptides having the aminoacid sequence shown below (SEQ ID NO: 6), ##STR9## or the amino acidsequence shown below (SEQ ID NO: 8). ##STR10##

Examples of the gene which codes for these polypeptides include thosecontaining the DNA sequence shown below (SEQ ID NO: 6), ##STR11## or theDNA sequence shown below (SEQ ID NO:8). ##STR12##

The polypeptide of the present invention, which has an epitope containedin the mite allergen derived from mite body, or the polypeptide havingan epitope which can be regarded as immunologically identical to saidepitope, is exemplified above. Although both of them can be used as anactive ingredient of a pharmaceutical composition for mite allergicdiseases or diagnostic reagent for mite allergic diseases, thepolypeptide of SEQ ID NO:8 is preferably used since it is more highlyantigenic.

The pharmaceutical composition for the treatment of mite allergicdiseases of the present invention contains as an active ingredient thepurified recombinant mite allergen, mite allergen fragment orepitope-containing polypeptide described above, and is used to treatvarious mite allergic diseases. Here, mite allergic diseases include allallergic diseases caused by the specific antigen of mites, such asatopic bronchial asthma, allergic rhinitis, allergic conjunctivitis andallergic dermatitis.

The pharmaceutical composition for the treatment of mite allergicdiseases of the present invention is not subject to limitation withrespect to the method of its preparation. For example, the recombinantmite allergen purified by the method described above or the miteallergen fragment or epitope-containing polypeptide purified inaccordance with the methods described above is dried and collected in apowder form and used as a hyposensitization therapeutic agent for miteallergic diseases. In hyposensitization therapy, the pharmaceuticalcomposition for mite allergic diseases of the present invention may beused as such or in the form of a formula preparation prepared by addingas necessary a commonly used adjuvant or various additives such as astabilizer, excipient, dissolution aid, emulsifier, buffer, soothingagent, preservative and colorant by a conventional method. For example,recombinant mite allergen purified in a powder form is dissolved in aphenol-containing saline, and this solution is used as a stock solutionof an antigen for hyposensitization therapy.

The pharmaceutical composition of the present invention thus obtainedcomprises as the active ingredient a pharmaceutically effective amountof the recombinant mite allergen and at least one pharmaceuticallyacceptable carrier or diluent.

The pharmaceutical composition for mite allergic diseases of the presentinvention can be administered by ordinary routes such as peroral,intracutaneous, subcutaneous, intramuscular and intraperitonealinjection. Moreover, it can be used as percutaneous or permucosal drugssuch as troches, sublingual tablets, eye drops, intranasal spray,poultices, creams and lotions.

As for the dosage and administration frequency of the pharmaceuticalcomposition for mite allergic diseases of the present invention, theyare appropriately selected according to the route of administration,symptoms and other conditions so that the dosage does not exceed about20 μg per administration for an adult, with an administration frequencyof about one time weekly.

The pharmaceutical composition for mite allergic diseases of the presentinvention is useful not only as a therapeutic agent but also as apreventive agent for mite allergic diseases. Since the pharmaceuticalcomposition for mite allergic diseases of the present invention is freefrom anaphylaxis inductive action, it can be safely used for humans.

The diagnostic reagent for mite allergic diseases of the presentinvention is used as a skin reaction diagnostic reagent for miteallergic diseases and as a titrating reagent for the diagnosis of miteallergy.

When used as a skin reaction diagnostic reagent, the diagnostic reagentfor mite allergic diseases of the present invention is prepared by aconventional method from the recombinant mite allergen purified by themethods described above or the mite allergen fragment orepitope-containing polypeptide purified in accordance with the methodsdescribed above. For example, the recombinant mite allergen is dried toa powder form, which is dissolved in a phenol-containing saline and usedin dilution. The use as a skin reaction diagnostic reagent is inaccordance with a conventional method.

Similarly, when used as a titrating reagent for the diagnosis of miteallergy, the diagnostic reagent for mite allergy diseases is prepared bya conventional method. For example, the recombinant mite allergen isappropriately dissolved in Hanks' solution and used after dilution as areagent for histamine release titration. This method is carried outnormally by the following procedure.

Blood of a mite allergy patient and a blood cell fraction obtained fromthis blood by centrifugation are suspended in a buffer to yield a bloodcell suspension. A given amount of this suspension is titrated using therecombinant mite allergen, a titrating reagent, and the amount ofhistamine released from basophiles (a kind of leukocyte) in response toallergen stimulation is determined by HPLC [Arerugi, 37, 725 (1988)].

In this histamine release titration, the amount of histamine released iscalculated from the 50% level (inflexion point of the titration curve)of the maximum release. This titration has two features. (1) Thepatient's allergen sensitivity is measured directly from the titer ofthe blood cell suspension. (2) After pre-reacting blood plasma andrecombinant mite allergen, the value obtained by titrating the bloodcell suspension with the reaction solution (blood titration curve value)is usually higher than the value obtained by titrating the blood cellsuspension with the recombinant mite allergen (blood cell suspensiontitration curve value). This is because the blood plasma contains an IgGantibody (blocking antibody) capable of allergen neutralization.

Therefore, the blocking antibody titer can be obtained from the degreeof shift of the blood titration curve from the blood cell suspensiontitration curve. On the basis of the allergen sensitivity and thisblocking antibody titer, accurate diagnosis of mite allergy is feasible.This histamine release titration test is useful to monitor the effect ofhyposensitization therapy.

EXAMPLES

The present invention is hereinafter described in more detail by meansof the following examples, but the invention is not limited by theseexamples.

EXAMPLE 1 Extraction of total mite RNA

6 g of live mite bodies obtained by cultivating Dermatophagoides farinaeby a conventional method, together with 10 g of quartz sand, in 200 mlof a solution of 5.5M guanidine isocyanate (produced by KatayamaKagaku), was ground in a mortar and centrifuged. The resultingsupernatant was repeatedly taken in and out with a 50-ml syringeequipped with an 18G injection needle to partially cleave the DNA. Afteradditional centrifugation, the supernatant was layered on a solution ofcaesium trifluoroacetate (produced by Katayama Kagaku) in a ratio of 16ml of the former to 17 ml of the latter and subjected to densitygradient centrifugation at 85000× g for 24° C. (15° C., HITACHI SCP55Hswing PRS-27-2), and the total RNA fraction forming a pellet on the tubebottom was recovered.

This total RNA fraction was dissolved in a solution of 4.0M guanidineisocyanate and precipitated with ethanol to yield 1 ml of a solution ofTE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA).

EXAMPLE 2 Separation of mite allergen Poly(A) mRNA

The TE solution of this total mite RNA was heated at 65° C. for 10minutes and rapidly cooled, after which an equal amount of 1M NaCl wasadded thereto, and the mixture was applied to an oligo (dT) cellulosecolumn (column volume 0.5 ml, produced by Boehringer Mannheim)pre-equilibrated with a solution of STE (10 mM Tris-HCl, pH 7.5, 1 mMEDTA, 0.5M NaCl). After the effluent was recycled to the column, thecolumn was washed with a 2.5-fold volume of STE solution. After columnwashing, poly(A) mRNA was eluted with TE solution. Here, 0.2 mlfractions were taken, and the fractions found to contain RNA by anethidium bromide spot test were pooled. This column purification wasconducted again, and the RNA was recovered by ethanol precipitation fromthe pooled fraction to yield a poly(A) mRNA fraction. The yield wasdetermined to be about 20 μg in a spot test together with knownconcentrations of serial dilutions of the RNA solution.

EXAMPLE 3 Synthesis of mite allergen cDNA

Next, in the presence of 5 μg of the poly(A) mRNA as a template, a cDNAhaving a 13 mer linker at both ends and containing an EcoR I site wassynthesized using a cDNA synthesis kit (produced by Pharmacia) inaccordance with the instruction manual.

EXAMPLE 4 Preparation of mite allergen cDNA library

A one-fifth amount of the cDNA was mixed with 1 μg of EcoR I-digested λgt11 (produced by Stratagene Cloning System). After concentration withethanol, this mixture was reacted with 200 units of T4 DNA ligase at 12°C. for 15 hours, whereby the cDNA was inserted into λ gt11 at the EcoR Isite. From this reaction mixture, a cDNA library was prepared using anin vitro packaging kit (Giga pack PLUS, produced by Stratagene CloningSystem) in accordance with the instruction manual. After two cycles ofthis library preparation procedure, a total of 41000 pfu (plaque formingunit) of cDNA was prepared from 2 μg of poly(A) mRNA.

EXAMPLE 5 Cloning of mite allergen cDNA

The cDNA-inserted λ gt11 phage was suspended in a solution of SM (1MTris-HCl, pH 7.5, 0.1M NaCl, 10 mM MgSO₄, 2% gelatin). This suspension,together with overnight-incubated E. coli Y1090 (produced by StratageneCloning System), was subjected to shaking culture at 37° C. for 30minutes to infect the host with the phage. The resulting culture wasspread over plates of LB agar medium [1% Bacto tryptone, 0.5% Bactoyeast extract, 1.5% Bacto agar (all produced by Difco Laboratories),0.5% NaCl] so that about 2000 plaques per plate were formed. Aftercultivation at 42° C. for 3 hours, the medium was covered with anitrocellulose membrane (Hibond-C, produced by Amersham) previouslydried after immersing in 10 mM isopropyl-1-thio-β-D-galactoside (IPTG),followed by additional cultivation at 37° C. for 3 hours and transfer ofthe inductively synthesized β-galactosidase-fused protein onto thenitrocellulose membrane. After blocking for overnight in a TBS solution(10 mM Tris-HCl, pH 8.0, 0.9% NaCl) containing 2% BSA, thenitrocellulose membrane was reacted with a rabbit anti-mite-body antigenserum (100-fold dilution in TBS) for 1 hour and then with aperoxidase-coupled goat anti-rabbit IgG antibody (produced by Cappel,2000-fold dilution in TBS) for 1 hour, and spots were visualized in aTBS solution containing 0.4 mg/ml diaminobenzidine tetrahydrochloride inthe presence of 0.01% hydrogen peroxide. The rabbit anti-mite-bodyantigen serum was obtained by suspending mite bodies from a whole miteculture with saturated saline and PBS, washing the mite bodies with PBS,grinding them and using the ground product as an immunogen for boosterimmunization of rabbits once weekly for 10 weeks. The plaque whichcorresponded to the position of the brown spot was picked up, suspendedin SM and stored as a positive clone.

EXAMPLE 6 Expression of fused protein using λ gt11 phage vector

The positive clone obtained by immunological screening in Example 5,together with 50 μg of an overnight culture broth of E. coli Y1090, wassubjected to shaking culture in 3 ml of an LB liquid medium containing100 μg of 100 mM IPTG at 37° C. for 8 hours, and the resulting culturesupernatant was dialyzed against distilled water and lyophilized toyield a sample for western blot.

SDS-PAGE was carried out in accordance with the method of Laemmli et al.[Nature, 227, 680-685 (1970)]. A 2 mg sample was electrophoresed underconditions of a polyacrylamide gel concentration of 5% and a constantelectric current of 15 mA. Then, the sample was transferred from theacrylamide gel to Immobilon membrane (pore size 0.45 μm, produced byMILLIPORE) by electrophoresis in an SDS-running buffer (192 mMglycine-25 mM Tris, pH 8.3, 0.1% SDS, 20% methanol) at 8 to 10 V/cm for2 hours. This Immobilon membrane was subjected to protein staining withAuro Dye (produced by JANSSEN Life Sciences Products) and immunologicalstaining with a pool serum of a mite allergy patient, rabbitanti-mite-body antigen serum and rabbit anti-β-galactosidase antibody(produced by 5 Prime →3 Prime Inc.), and the mite allergen fused proteinwas detected (FIG. 1).

EXAMPLE 7 Construction of recombinant plasmid

After recovery from the λ gt11 phage clone inserting the cDNA whichcodes for the mite allergen-β-galactosidase fused protein which reactedwith these antisera, the DNA was digested with EcoR I. This digestionproduct was subjected to agarose gel electrophoresis, and the cDNA chainlength was determined to be about 1.2 kbp (including the linker)(indicated by arrow in FIG. 2). Next, the λ gt11 phage DNA inserted thiscDNA was digested with Kpn I-Sac I. The obtained fragment (about 3.3kbp) was ligated to the plasmid vector pUC18 (produced by Takara Shuzo)at the Kpn I-Sac I site and transformed into E. coli JM109 (produced byTakara Shuzo) in accordance with the method of Hanahan [DNA Cloning,vol. 1, Glover, D. M. ed., pp. 109-136, IRL Press (1985)] (FIG. 3).

The obtained recombinant plasmid was named pAK 1, which was used to drawa restriction enzyme map (1144 bp, not including the linker) (FIG. 4).

EXAMPLE 8 Determination of nucleotide sequence of mite allergen cDNA

The EcoR I digestion fragment of the plasmid pAK 1 was ligated to thesame plasmid vector pUC18 at the EcoR I site and transformed into E.coli JM109 in the same manner (FIG. 5).

The obtained recombinant plasmid was named pAKE 1, which was used todraw a restriction enzyme map (940 bp, not including the linker) (FIG.4). Next, a single-stranded DNA was prepared by a conventional methodfrom the culture supernatants of transformants of the phage vectorsM13mp18 and mp19 (produced by Takara Shuzo) containing various fragmentsobtained by digesting the recombinant plasmids pAK 1 and pAKE 1 withrestriction enzymes such as EcoR I and BamH I, and its partialnucleotide sequence was determined by the dideoxy nucleotide chaintermination method using Sequenase Version 2.0 (produced by Toyobo Ltd.)(SEQ ID NO:1).

Through repeated investigation of the nucleotide sequence by the samemethod, a nucleotide sequence of about 1.1 kbp (not including thelinker) containing the above-mentioned nucleotide sequence (SEQ ID NO:1)was determined (SEQ ID NO:2).

EXAMPLE 9 Construction of high expression vector

The EcoR I digestion fragment (940 bp) of the recombinant plasmid pAKE 1was subjected to agarose gel electrophoresis and extracted using theGeneclean II kit (produced by BIO 101). This extract was ligated to thevector pUEX 2 (produced by Amersham), previously cleaved by EcoR Idigestion, so that the frame fit to the direction of translation (FIG.6).

EXAMPLE 10 Expression as fused protein

The recombinant plasmid obtained in Example 9 was named pAEX201, whichwas transformed into E. coli pop2136 (produced by Amersham).

This transformant was subjected to shaking culture in 500 ml of an SBAliquid medium (2.4% Bacto yeast extract, 1.2% Bacto polypeptone, 0.5%glycerol, 0.1M potassium phosphate, pH 7.5, 50 mg/ml ampicillin) at 28°C. until the OD value at 600 nm became 0.6. After an equal amount of SBAmedium maintained at 56° C. was added, shaking culture was continued at42° C. for additional 90 minutes. An appropriate amount of fresh SBA wasadded at appropriate times so that the OD value was maintained between0.6 and 1.0 during the inductive synthesis of fused protein by thistemperature shift to 42° C. After completion of the cultivation, theculture broth was centrifuged. Cells were harvested, washed with sterilewater and were suspended in 10 ml of a solution containing proteaseinhibitors [0.1M Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 mMphenylmethanesulfonyl fluoride, 1 mM iodoacetic acid, 5 mM1,2-epoxy-3-(p-nitrophenoxy)-propane]. This suspension was subjected to10 cycles of 10-20 kHz ultrasonication for 1 minute to disrupt thecells. After centrifugation, the pellet was suspended in 10 ml of a0.02% SDS solution. After re-centrifugation, the pellet-formingmembrane-localized protein was completely dissolved in 10 ml of a 2% SDSsolution. The lyophilized product of this extract was analyzed bySDS-PAGE. The results are shown in FIG. 7.

EXAMPLE 11 Purification of recombinant mite allergen fused protein

The yield of the lyophilization product of the extract was 122 mg from500 ml of the culture broth. Five mg of the lyophilization product wasdissolved in 500 μl of water, and this solution was applied to a coupleof columns of Ultrogel AcA 44 (produced by LKB, 1.4×50 cm)+AG 11A8(produced by Bio-Rad, 1.4×14 cm) and developed using deionized water asa developing solvent at a flow rate of 10 ml/hr, and the fraction elutedin void volume was collected (FIG. 8). The yield of the lyophilizationproduct of this fraction was 0.6 mg. The lyophilization product was thenpassed through an anti-β-galactosidase antibody immobilized affinitycolumn and then purified using an anti-mite-body antibody immobilizedaffinity column. The anti-β-galactosidase antibody immobilized affinitycolumn was prepared by immobilizing an anti-β-galactosidase antibody(produced by 5Prime→3Prime Inc.) to tresyl GM gel (produced by KuritaWater Industries Ltd.). The anti-mite-body antibody immobilized affinitycolumn was prepared by fractionating the rabbit anti-mite-body antigenserum described in Example 5 with ammonium sulfate, then purifying theammonium sulfate fraction using a column of protein A and immobilizingit onto tresyl GM gel.

EXAMPLE 12 Histamine release test using purified fused protein

A solution of the purified antigen (1 mg/ml) was diluted to anappropriate volume. To 200 μl of this diluted solution, 200 μl of asuspension of blood cells from a mite asthma patient, washed with Hanks'solution, was added, and reaction was carried out at 37° C. for 30minutes. After centrifugation at 1400 rpm for 10 minutes, 200 μl of thesupernatant was taken. To the supernatant, 10 μl of 60% perchloric acidwas added, and this mixture was vigorously stirred and centrifuged at10000 rpm for 10 minutes to remove the protein. 150 μl of thissupernatant was subjected to HPLC to determine the amount of histaminein 100 μl of the supernatant. The total amount of histamine wasdetermined in the same manner as above except that blood cell removal bycentrifugation was not conducted.

The obtained results are shown in FIG. 9, in which antigen concentrationis plotted on the abscissa and the percent ratio of the releasedhistamine [(amount of released histamine/total amount of histamine)×100]is plotted on the ordinate. Comparing the concentration at the inflexionpoint reveals that this mite allergen fused protein reacted at aconcentration lower by over 200 times than do the crude mite bodyantigen.

EXAMPLE 13 Preparation of recombinant mite allergen fragment

Using pUEX 2 inserting the 970 bp EcoR I fragment of cDNA, deletion wascarried out from the downstream of cDNA with Exonuclease III (producedby Takara Shuzo, Deletion kit for Kilo-Sequence). As a result, 10 kindsof cDNA with a deleted nucleotide chain were obtained as shown in FIG.10.

These deleted cDNA fragments were each transformed into E. coli, and thecells were grown on a nitrocellulose membrane at 28° C. After subsequentinductive expression at 42° C. for 2 hours and lysis with SDS at 100°C., the cell component adsorbed onto the nitrocellulose membrane wasvisualized for antigen activity by the enzyme-linked immunosorbentmethod using an anti-mite antigen serum.

The results are shown in FIG. 10. It was found that the activity whichremained at the 6th spot from the left in the figure after deletion upto about 400 bp mostly disappeared by deletion up to about 270 bp at the7th spot and the activity completely disappeared by deletion up to about170 bp at the 8th spot.

This finding suggested that the antibody avidity required at least aregion about 170 bp to about 270 bp, about 270 bp to about 400 bp, orabout 170 bp to about 400 bp from the upstream; the recombinant miteallergen fragment containing the amino acid sequence of this regioncould be used as an active ingredient of therapeutic agents anddiagnostic reagents for mite allergic diseases.

EXAMPLE 14 Preparation of epitope-containing polypeptide

On the basis of the results of Example 13, epitope regions in the170-400 bp region were identified as shown in FIG. 11 by adjusting thereaction time of Exonuclease II (produced by Takara Shuzo, Deletion kitfor Kilo-Sequence) in the same manner as in Example 13. It was foundthat two regions of 181-246 bp and 343-378 bp were essential as epitopeparts. The nucleotide sequence to the enzyme cleavage point wasdetermined using TAQuence (produced by Toyobo Ltd.). Polypeptidescontaining the amino acid sequences (SEQ ID NO:3 and SEQ ID NO:4)deduced from these nucleotide sequences can easily be synthesized by aconventional method using, for example, a peptide synthesizer producedby Applied Biosystems.

EXAMPLE 15 Preparation of therapeutic agent for mite allergic diseases

The purified allergen active component is dried, collected in a powderform and used as a hyposensitization therapeutic agent for mite allergypatients.

The allergen active component is dissolved in a 0.9% saline containing0.5% phenol to a final concentration of 1 mg/ml to yield a stocksolution of an antigen for hyposensitization therapy.

EXAMPLE 16 Preparation of diagnostic reagent for mite allergic diseases

The purified allergen active component is dried, collected in a powderform and used as a skin reaction diagnostic reagent for mite allergicdiseases and as a titrating reagent for the diagnosis of mite allergy.

The skin reaction diagnostic reagent is prepared by 200,000-folddiluting the allergen active component in a 0.9% physiological salinecontaining 0.5% phenol.

The titrating reagent for the diagnosis of mite allergy was prepared bydissolving the allergen active component in Hanks' buffer solution at aconcentration of 1 mg/ml to yield a stock solution of a reagent forhistamine release titration and diluting the stock solution.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 249 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Dermatophagoides farinae                                       (F) TISSUE TYPE: mite body                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..249                                                          (D) OTHER INFORMATION: /product="mite body allergen"                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TTTGTCATGAAACGAGAACCATTGCGATTCAGAGACATCACTGTCGAA48                            PheValMetL ysArgGluProLeuArgPheArgAspIleThrValGlu                             151015                                                                        GGAAACGAAAATGCCTATATCAAAAATGGCAAACTTCATTTGTCGCTT96                            GlyAsnGlu AsnAlaTyrIleLysAsnGlyLysLeuHisLeuSerLeu                             202530                                                                        ATGGATCCGTCAACATTGAGTTTAGTCACGAAAGCCGATGGAAAAATC144                           MetAspProSer ThrLeuSerLeuValThrLysAlaAspGlyLysIle                             354045                                                                        GACATGACAGTAGACTTGATATCGCCAGTCACAAAACGTGCATCGTTG192                           AspMetThrValAspLe uIleSerProValThrLysArgAlaSerLeu                             505560                                                                        AAAATTGATTCAAAGAAATACAACCTTTTCCATGAAGGTGAATTGAGT240                           LysIleAspSerLysLysTyrAsnL euPheHisGluGlyGluLeuSer                             65707580                                                                      GCATCGATC249                                                                  AlaSerIle                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 83 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       PheValMetLysArgGluProLeuArgPheArgAspIleThrValGlu                              1510 15                                                                       GlyAsnGluAsnAlaTyrIleLysAsnGlyLysLeuHisLeuSerLeu                              202530                                                                        MetAspProSerThrLeuSerLeuValThrLysAlaAspGly LysIle                             354045                                                                        AspMetThrValAspLeuIleSerProValThrLysArgAlaSerLeu                              505560                                                                        LysIleAspSerLysL ysTyrAsnLeuPheHisGluGlyGluLeuSer                             65707580                                                                      AlaSerIle                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1023 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Dermatophagoides farinae                                        (F) TISSUE TYPE: mite body                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1023                                                         (D) OTHER INFORMATION: /product="mite body allergen"                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TTTGTCATGAAACGAGAACCAT TGCGATTCAGAGACATCACTGTCGAA48                           PheValMetLysArgGluProLeuArgPheArgAspIleThrValGlu                              151015                                                                        GGAAACGAAAATGCCTATATC AAAAATGGCAAACTTCATTTGTCGCTT96                           GlyAsnGluAsnAlaTyrIleLysAsnGlyLysLeuHisLeuSerLeu                              202530                                                                        ATGGATCCGTCAACATTGAGTTTA GTCACGAAAGCCGATGGAAAAATC144                          MetAspProSerThrLeuSerLeuValThrLysAlaAspGlyLysIle                              354045                                                                        GACATGACAGTAGACTTGATATCGCCAGT CACAAAACGTGCATCGTTG192                          AspMetThrValAspLeuIleSerProValThrLysArgAlaSerLeu                              505560                                                                        AAAATTGATTCAAAGAAATACAACCTTTTCCATGAAG GTGAATTGAGT240                          LysIleAspSerLysLysTyrAsnLeuPheHisGluGlyGluLeuSer                              65707580                                                                      GCATCGATCGTAAACCCACGATTGTCATGGCAT CAATACACGAAACGC288                          AlaSerIleValAsnProArgLeuSerTrpHisGlnTyrThrLysArg                              859095                                                                        GATTCTCGTGAATACAAGAGTGATGTAGAACTA TCGTTGCGATCGTCG336                          AspSerArgGluTyrLysSerAspValGluLeuSerLeuArgSerSer                              100105110                                                                     GACATTGCTCTCAAGATTACGATGCCTGATTATAA TTCGAAAATTCAT384                          AspIleAlaLeuLysIleThrMetProAspTyrAsnSerLysIleHis                              115120125                                                                     TATTCACGACAAGGTGATCAAATCAACATGGACATCGATG GTACATTG432                          TyrSerArgGlnGlyAspGlnIleAsnMetAspIleAspGlyThrLeu                              130135140                                                                     ATCGAAGGTCATGCACAAGGAACCATCAGAGAAGGTAAAATCCACATT 480                          IleGluGlyHisAlaGlnGlyThrIleArgGluGlyLysIleHisIle                              145150155160                                                                  AAAGGTAGACAAACTGATTTCGAGATCGAATCCAACTACCGATAC GAA528                          LysGlyArgGlnThrAspPheGluIleGluSerAsnTyrArgTyrGlu                              165170175                                                                     GATGGCAAACTAATCATCGAACCGGTCAAGAGTGAAAATGGCAA ATTG576                          AspGlyLysLeuIleIleGluProValLysSerGluAsnGlyLysLeu                              180185190                                                                     GAAGGCGTTCTTTCCCGTAAGGTGCCATCACATCTGACACTAGAAA CA624                          GluGlyValLeuSerArgLysValProSerHisLeuThrLeuGluThr                              195200205                                                                     CCACGAGTCAAGATGAATATGAAATATGATCGATATGCACCAGTCAAA 672                          ProArgValLysMetAsnMetLysTyrAspArgTyrAlaProValLys                              210215220                                                                     GTGTTCAAATTGGATTATGATGGCATCCACTTCGAGAAACATACCGAT720                           ValP heLysLeuAspTyrAspGlyIleHisPheGluLysHisThrAsp                             225230235240                                                                  ATTGAATACGAACCTGGCGTTCGATACAAGATCATCGGCAATGGAAAA768                            IleGluTyrGluProGlyValArgTyrLysIleIleGlyAsnGlyLys                             245250255                                                                     CTCAAGGATGATGGCCGCCACTATTCTATCGATGTGCAAGGTATTCCA816                            LeuLysAspAspGlyArgHisTyrSerIleAspValGlnGlyIlePro                             260265270                                                                     CGCAAAGCATTCAATCTGGACGCTGACTTGATGGATTTCAAACTGAAA864                           Ar gLysAlaPheAsnLeuAspAlaAspLeuMetAspPheLysLeuLys                             275280285                                                                     GTGAGCAAGCCAGAAGATAGCAATAAAGCTCAATTCAGCTACACATTC912                           ValSerL ysProGluAspSerAsnLysAlaGlnPheSerTyrThrPhe                             290295300                                                                     AACGAATATACCGAGACCGAAGAATATGAATTCGATCCACATCGTGCC960                           AsnGluTyrThrGlu ThrGluGluTyrGluPheAspProHisArgAla                             305310315320                                                                  TATTATGTTAATTGGTTGAGTTCCATTCGCAAATACATCCAGAATTTC1008                          TyrTyrValAsn TrpLeuSerSerIleArgLysTyrIleGlnAsnPhe                             325330335                                                                     ATCGTCGAAGACAAC1023                                                           IleValGluAs pAsn                                                              340                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 341 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       PheValMetLysArgGluProLeuArgPheArgAspIleThrValGlu                               151015                                                                       GlyAsnGluAsnAlaTyrIleLysAsnGlyLysLeuHisLeuSerLeu                              202530                                                                        MetAspProSer ThrLeuSerLeuValThrLysAlaAspGlyLysIle                             354045                                                                        AspMetThrValAspLeuIleSerProValThrLysArgAlaSerLeu                              5055 60                                                                       LysIleAspSerLysLysTyrAsnLeuPheHisGluGlyGluLeuSer                              65707580                                                                      AlaSerIleValAsnProArgLeuSerTrpHisGlnTyr ThrLysArg                             859095                                                                        AspSerArgGluTyrLysSerAspValGluLeuSerLeuArgSerSer                              100105110                                                                     A spIleAlaLeuLysIleThrMetProAspTyrAsnSerLysIleHis                             115120125                                                                     TyrSerArgGlnGlyAspGlnIleAsnMetAspIleAspGlyThrLeu                              130 135140                                                                    IleGluGlyHisAlaGlnGlyThrIleArgGluGlyLysIleHisIle                              145150155160                                                                  LysGlyArgGlnThrAspPheGluIle GluSerAsnTyrArgTyrGlu                             165170175                                                                     AspGlyLysLeuIleIleGluProValLysSerGluAsnGlyLysLeu                              180185 190                                                                    GluGlyValLeuSerArgLysValProSerHisLeuThrLeuGluThr                              195200205                                                                     ProArgValLysMetAsnMetLysTyrAspArgTyrAlaProValLys                               210215220                                                                    ValPheLysLeuAspTyrAspGlyIleHisPheGluLysHisThrAsp                              225230235240                                                                  IleGluTyrGluProG lyValArgTyrLysIleIleGlyAsnGlyLys                             245250255                                                                     LeuLysAspAspGlyArgHisTyrSerIleAspValGlnGlyIlePro                              260 265270                                                                    ArgLysAlaPheAsnLeuAspAlaAspLeuMetAspPheLysLeuLys                              275280285                                                                     ValSerLysProGluAspSerAsnLysAlaGlnPheSer TyrThrPhe                             290295300                                                                     AsnGluTyrThrGluThrGluGluTyrGluPheAspProHisArgAla                              305310315320                                                                  TyrTy rValAsnTrpLeuSerSerIleArgLysTyrIleGlnAsnPhe                             325330335                                                                     IleValGluAspAsn                                                               340                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Dermatophagoides farinae                                        (F) TISSUE TYPE: mite body                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..66                                                           (D) OTHER INFORMATION: /product="mite body allergen"                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ATGACAGTAGACTTGATATCGCCAGTCACAAAACGTGCATCGTTGAAA48                            MetThrValAspLeuIleSerProValThrLysArgAlaSerLeuLys                              1510 15                                                                       ATTGATTCAAAGAAATAC66                                                          IleAspSerLysLysTyr                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetThrValAspLeuIleSerProValThrLysArgAlaSerLeuLys                              151015                                                                        IleAspSerLysLysTyr                                                            2 0                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Dermatophagoides farinae                                        (F) TISSUE TYPE: mite body                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..36                                                          (D) OTHER INFORMATION: /product="mite body allergen"                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GATGTAGAACTATCGTTGCGATCGTCGGACATTGCT36                                        AspValGluLeuSerLeuArgSerSerAspIleAla                                          15 10                                                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AspValGluLeuSerLeuArgSerSerAspIleAla                                          15 10                                                                     

What is claimed is:
 1. A substantially pure recombinant mite allergenobtainable by expression of a mite-body-derived gene fragment, whichcontains the partial amino acid sequence shown in SEQ ID NO:2: ##STR13##2. A substantially pure recombinant mite allergen obtainable byexpression of a mite-body-derived gene fragment, which contains thepartial amino acid sequence shown in SEQ ID NO:4: ##STR14##
 3. Arecombinant mite allergen according to claim 1 wherein said genefragment is derived from Dermatophagoides farinae.
 4. A recombinant miteallergen according to claim 1, wherein the molecular weight of saidallergen is about 40,000 as determined by SDS-PAGE.
 5. A recombinantmite allergen according to claim 1, wherein the total nucleotide chainlength of the cDNA of said allergen is about 1.2 kbp as determined byagarose electrophoresis, and which is obtainable by expression of a genefragment containing the restriction enzyme map shown in FIG.
 4. 6. Afused recombinant mite allergen, wherein the recombinant mite allergenof claim 1 is fused with another protein.
 7. A fused recombinant miteallergen according to claim 6, wherein said protein is β-galactosidase.8. A substantially pure mite allergen fragment containing at least anamino acid sequence encoded in the region of about 170 bp to about 270bp, about 270 bp to about 400 bp, or about 170 bp to about 400 bp fromthe translation start in the restriction map shown in FIG.
 10. 9. Asubstantially pure polypeptide having an epitope contained in an aminoacid sequence encoded by a DNA fragment as shown in the restriction mapshown in FIG. 10, said DNA fragment being a cDNA obtained fromDermatofagoides farinae, said DNA fragment being selected from the groupconsisting of 170 bp to 270 bp from the start of translation, 270 bp to400 bp from the start of translation and 170 to 400 bp from the start oftranslation.
 10. A polypeptide according to claim 9, containing theamino acid sequence shown in SEQ ID NO:6:Met Thr Val Asp Leu Ile Ser ProVal Thr Lys Arg Ala Ser Leu Lys Ile Asp Ser Lys Lys Tyr.
 11. Apolypeptide according to claim 9, containing the amino acid sequenceshown in SEQ ID NO:8:Asp Val Glu Leu Ser Leu Arg Ser Ser Asp Ile Ala.12. A diagnostic reagent for mite allergic diseases, which comprises asthe active ingredient a diagnostically effective amount of therecombinant mite allergen of claim
 1. 13. A diagnostic reagent for miteallergic diseases, which comprises as the active ingredient adiagnostically effective amount of the mite allergen fragment of claim8.
 14. A diagnostic reagent for mite allergic diseases, which comprisesas the active ingredient a diagnostically effective amount of thepolypeptide of claim
 10. 15. A substantially pure recombinant miteallergen, wherein said recombinant mite allergen has the amino acidsequence shown in SEQ. ID. No. 1 and is produced by a process comprisingthe steps of:(a) expressing a fusion gene composed of amite-body-derived gene ligated to a gene encoding at least a secondprotein; (b) collecting a fusion protein composed of a mite-body-derivedallergen and said second protein obtained from step (a); (c) separatingsaid second protein from said mite-body-derived allergen; (d) collectingsaid mite-body-derived allergen.
 16. A substantially pure recombinantmite allergen, wherein said recombinant mite allergen has the amino acidsequence shown in SEQ. ID. No. 2 and is produced by a process comprisingthe steps of:(a) expressing a fusion gene composed of amite-body-derived gene ligated to a gene encoding at least a secondprotein; (b) collecting a fusion protein composed of a mite-body-derivedallergen and said second protein obtained from step (a); (c) separatingsaid second protein from said mite-body-derived allergen; (d) collectingsaid mite-body-derived allergen.